Pain and opioid antinociception are known to manifest sexual dimorphism. However, a male/female dichotomy in opioid tolerant and withdrawal mechanisms has not been demonstrated or assiduously investigated. We have recently demonstrated that tolerance mechanisms utilized are dynamic and pliable [1]. Since ovarian sex steroids modulate multiple parameters of cell functionality, we hypothesize that adaptations elicited by chronic morphine would manifest sexual dimorphism. This will be tested by assessing sex-dependence of adaptations of the m-opioid receptor (MOR)-coupled regulation of spinal endomorphin (EM) 2 release to opioid tolerance and withdrawal. We elected to study spinally released EM2 since it has sexually dimorphic characteristics [[and is the predominant spinal EM]]. Our initial observations underscore the relevance of sex-based tolerance and withdrawal adaptations to spinal EM2 release homeostasis. Predominance of MOR Gs facilitation of EM2 release was manifest in in vitro withdrawn spinal tissues of males, but not females. This suggests that in females, spinal EM2 is not recruited to compensate for the abrupt cessation of MOR activation, as they appear to be in males. Preliminary data also indicate a male-female dichotomy in spinal tolerance adaptations. The abolishment vs. partial loss of MOR-coupled inhibition of EM2 release in tolerant spinal tissue of females and males, respectively, suggests that preservation of normative EM2 release from tolerant spinal tissue is sex- dependent. Elucidation of cellular tolerance [[and withdrawal]] mechanisms that influence spinal EM2 release will enable assessment of the functional relevance of selected chronic morphine-induced biochemical adaptations within a closed loop physiological context. This will enhance the translational utility of sexual dimorphic adaptational mechanisms that are revealed. A main underlying hypothesis of this proposal is that [[(1)]] long-term systemic morphine treatment [[(tolerance)]] and [[(2)]] its in vitro withdrawal elicits sex- dependent adaptations that make use of plasticity inherent in G protein-coupled signaling (receptor pleiotropy) to maintain normative spinal EM2 release. This involves changes in MOR coupling from predominantly Gi/Go to Gs. The overarching rubric encompassing each of the four Aims is that MOR-coupled regulation of EM2 release exhibits physiological state-dependency, enabling the sensory MOR system to accommodate changing physiological demand. Aims 1 and 2 will investigate, using selective in vivo G protein ablation, the relevance of facilitative MOR signaling to spinal EM2 release during opioid withdrawal and tolerance, respectively. Aim 3 will define the biochemical substrates causally associated with the emergence of MOR-coupled facilitation of EM2 release in opioid [[tolerant vs. withdrawn]] spinal tissue. Aim 4 will establish sexual dimorphism, and the relevance of sex steroids to its expression, of chronic morphine-induced adaptations identified in preceding Aims. Elucidation of sexual dimorphic dimensions of [[tolerance vs. withdrawal]] could profoundly influence their medical management and the pain control strategies employed in women as well as men.